The purpose of these studies is to establish a better understanding of the energy metabolism of biological tissues. Towards this goal, the laboratory concentrates on the use of screening approaches in proteomics and post-translational modifications. The following major findings were made over the last year: 1) We have continued to develop a non-destructive optical spectroscopy method using a center mounted integrating sphere and a rapid scanning spectroscopy system to monitor the redox sensitive chromophores of mitochondrial oxidative phosphorylation minimizing light scattering effects. Using this approach we have established characterized all of the redox chromophores in the mitochondria and begun to establish the regulation of reducing equivalent distribution within the network. We have described that both the activation, with calcium, and deactivation, ischemia reperfusion in intact heart, that all of the Complexes of oxidative phosphorylation are modified in concert. These data imply that the activity of these Complexes is orchestrated together in the intact mitochondria. The mechanisms responsible for the coordination of the Complex activities are still under investigation. Our working hypothesis is based on the fact that the mitochondria evolved from early symbiotic bacteria retaining many of bacteria protein synthesis processes and even DNA. We speculate that the signaling mechanisms within the mitochondria may also be closer to bacterial signaling systems than the more familiar eukaryotic systems. 2) To test the bacterial signaling hypothesis stated above, we have initiated studies on isolated bacteria believed to be closest to the mitochondrial origins, paracoccus denitrificans. Our initial studies have demonstrated that the respiratory rate, or ATP production rate, can be acutely modulated using the volume regulatory processes in these bacteria. Using this approach we have surprisingly demonstrated that the bacteria up-regulate metabolic capacity acutely with increases in work demand based on the increase in mitochondrial NADH with work transitions. This effect seems to parallel processes we have observed in the mammalian mitochondria. We are continuing to characterize the basic energy metabolism of paracoccus during growth and volume regulatory processes using our proteomic and non-invasive optical approaches. It is hoped that this simplified system will provide insights into the regulation of mitochondrial oxidative phosphorylation, in the coming year. 3) Our previous work on the regulation of oxidative phosphorylation has concentrated on isolated mitochondria that we have extrapolated to in vivo conditions. We are now moving our non-invasive optical studies of the chromophores of oxidative phosphorylation into the study of the isolated perfused heart. We have demonstrated that a homemade artificial oxygen delivery system, based on perflurocarbons, works in the perfused heart increasing oxygen delivery by more than 5 fold. Spectroscopic studies demonstrate that this increase in oxygen delivery to the classical perfused hearts of rat and rabbits increases the oxygenation of both the myoglobin as well as the mitochondria, based on the absorbance of cytochrome oxidase. These data suggest that using the classical saline perfused heart my introduce hypoxia in the perfused heart models. It is hoped that that this system will permit us to extrapolate our isolated mitochondria observations to a working intact tissue over the next year. 4) We completed our studies on cAMP and have reached the conclusion that mitochondrial matrix cAMP doe not impact the capability of the mitochondria to generate ATP nor change the redox poise of the cytochrome chain. We believe this make matrix cAMP a very unlikely candidate in the acute regulation of mitochondrial ATP production. 5) We demonstrated with Dr. Finkel and Murphy that knocking out the putative mitochondrial Ca transporter, MCU, had minimal cardiac performance effects and only impacted the peak performance of the skeletal muscle. This was consistent with our previous work on the allometry of cardiac energetics that demonstrated that the mouse heart was near maximal performance even at rest, not requiring the calcium activation system, while the skeletal muscle still had considerable dynamic range that required full metabolic activation at peak power. These data are consistent with other knockout studies in our lab over the last 2 years where we have removed the two major facilitated diffusion systems, myoglobin and creatine, and also only found effects at peak power in the skeletal muscles of the mice. These results suggest that major enzyme systems are present in mice that only support small increments in peal performance that might have significant evolutionary survival advantages for this small mammal.